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摘 要：目的：研究低氧和低氧训练对AMPKα2转基因小鼠骨骼肌CPT-1的mRNA和蛋白表达水平的影响，进而探讨低氧和低氧训练中AMPKα2调节脂肪酸氧化过程的可能机制。方法：健康两月龄C57BL/6J品系的野生小鼠和AMPKα2转基因小鼠，分别分为常氧安静组、低氧安静组和低氧训练组，共六组，每组10只。低氧组小鼠持续暴露于低氧房中（模拟海拔4000m高度，氧浓度12.3%），其中低氧训练组小鼠还要在此低氧房中进行跑台运动，12m/min，每天1h，持续2周。测定股四头肌 AMPKα2蛋白水平以及CPT-1的mRNA和蛋白表达情况。结果：2周的低氧和低氧训练对AMPKα2的蛋白表达没有显著的影响；低氧和低氧训练均显著提高了CPT-1的mRNA表达（P<0.05）；低氧对野生和AMPKα2转基因小鼠CPT-1的作用无差异；低氧训练组中，AMPKα2转基因小鼠的CPT-1 mRNA表达显著高于野生小鼠（P<0.05），与低氧安静组相比，低氧训练使转基因小鼠的CPT-1蛋白表达产生了显著增加（P<0.05），但是野生小鼠并无显著变化。结论：AMPKα2参与调节了低氧训练中骨骼肌CPT-1的表达水平，促进脂肪酸的氧化过程。
关键词：AMPKα2；CPT-1；低氧；低氧训练；骨骼肌
中图分类号：G804.7文献标识码：A文章编号：1007-3612(2011)01-0051-04
The Effects of Hypoxia and Hypoxia Training on the Expression of CPT1 in AMPKα2 Transgenic Mice Skeletal Muscle
YAO Lu，XIE Jin,XIE Jin，LI Songbo，ZHANG Ying
(Beijing Sport University, Beijing 100084, China)
Abstract:Objective: To study the effects of hypoxia and hypoxia training on the expressions of CPT1 mRNA and protein in AMPKα2 transgenic mice skeletal muscle. Furthermore, we explore the possible mechanism of AMPKα2 in regulating fatty acid oxidation during hypoxia and hypoxia training. Methods: Healthy C57BL/6J wildtype(WT) mice and AMPKα2 transgenic(TG) mice, two months old, were randomly divided into control groups, hypoxia groups and hypoxia training groups each.Hypoxia groups and hypoxia training groups were exposed in the hypoxia room the whole day for two weeks (simulation of 4000m altitude, oxygen concentration 12.3%), and meanwhile hypoxia training groups were trained to run on the treadmill (12 m/min, 1h/day). After that, quadriceps femoris muscle were removed, and then the expression of AMPKα2 protein and CPT1 mRNA and protein were measured. Results: Either hypoxia or hypoxia training had no effect on the expression of AMPKα2 protein; both hypoxia and hypoxia training significantly increased CPT1 mRNA expression(P<0.05); in hypoxia groups, there was no apparent difference in CPT1 expression between WT and TG mice; after two weeks hypoxia training, the expression of CPT1 mRNA of TG mice was significantly higher than that of WT mice(P<0.05) and compared with hypoxia groups, the expression of CPT1 protein of TG mice was higher in hypoxia training groups(P<0.05), but hypoxia training didn’t change that of WT mice. Conclusion: During hypoxia training, AMPKα2 is activated to regulate the expression level of CPT1 in skeletal muscle, so that it stimulates fatty acid oxidation.
Key words: AMPKα2; CPT1; hypoxia; hypoxia training; skeletal muscle
5’-腺苷酸活化的蛋白激酶（5’-AMP-activated protein kinase, AMPK）在调节和维持能量代谢平衡中发挥着非常重要的作用，被称为细胞的能量感受器。运动、缺血、缺氧等刺激都会造成细胞内的AMP/ATP比值升高，使 AMPK活化并作用于下游靶蛋白，开启ATP的合成途径，同时关闭多条ATP的分解途径。脂肪酸的β氧化是ATP再合成的重要途径之一，然而长链脂肪酸不能通过扩散的方式进入线粒体，肉碱棕榈酰转移酶-1（carnitine palmitoyl transferase1，CPT-1）位于线粒体外膜的外侧，是长链脂肪酸进入线粒体氧化的限速酶［1］。已有研究表明长期适当的运动训练能够增加AMPK和CPT-1的活性和表达水平［2-6］，但对于低氧和训练结合时AMPK和CPT-1表达水平的研究尚少。本实验旨在观察低氧和低氧训练对AMPKα2转基因小鼠骨骼肌CPT-1的mRNA和蛋白表达的影响，以探讨AMPKα2调节脂肪酸氧化的可能机制。
1 实验材料与方法
1.1 实验对象与分组 健康两月龄的C57BL/6J品系小鼠，野生小鼠（Wild-type，WT）和AMPKα2转基因小鼠（Transgenic，TG）各30只，体重(18±2)g。AMPKα2转基因小鼠由中国医学科学院实验动物研究所建立并繁育，全身高表达AMPKα2基因。所有小鼠均在北京体育大学动物房中饲养，温度20～25℃，相对湿度50°～70°，每天光照12 h，自由摄食和饮水，国家标准啮齿类动物饲料。
不同实验结果的差异性可能是由实验对象、取材部位、研究方法、低氧暴露模式（如氧浓度、低氧持续时间）等方面的原因造成的。
有学者指出长期低氧应激使骨骼肌纤维中的线粒体含量减少［16］，机体更加依赖糖类作为代谢底物［16,17］。在产生等量ATP的情况下，脂肪氧化的耗氧量多于糖代谢，所以低氧适应可能更多地通过增加糖的有氧氧化和糖酵解过程为机体提供能量［17］。因此，本实验中，低氧对野生和转基因小鼠CPT-1的作用无差异，可能与此有关。在低氧环境下AMPKα2的高表达，可能更多地体现在对糖代谢的调节上，而对脂肪氧化中的CPT-1的影响不大。目前，关于低氧对骨骼肌CPT-1影响的研究还很少，有待进一步深入研究。
3.2.2 低氧训练对AMPKα2转基因小鼠CPT-1表达的影响 运动可以促进脂肪酸氧化，改善对储备脂肪的利用，抑制脂肪组织的增长［18］。运动激活的AMPK将脂肪酸合成的限速酶——乙酰辅酶A羧化酶（acetyl-CoA carboxylase，ACC）磷酸化并抑制其活性，同时磷酸化丙二酰辅酶A脱羧酶（malonyl-CoA decarboxylase，MCD）使其活化，减少了丙二酰CoA的产生，解除对CPT-1的抑制，CPT-1的活性增强，进入线粒体的脂肪酸增多，促进β氧化。
人类肌肉活检和动物实验都发现耐力训练能够适应性地提高骨骼肌CPT-1的活性［3，4，19］。运动训练除了改善酶的活性，还可以引起表达水平的变化，对代谢的影响更为深刻［20］。每天以63%VO2max的强度蹬车1 h，连续运动9 d，CPT-1的mRNA表达显著提高［5］。张癑等发现，高脂饮食结合运动组大鼠骨骼肌中CPT-1的转录和翻译水平发生了显著的增加［6］。在本研究中，低氧训练组由于低氧和运动的双重刺激，使机体处于极大地应激状态。骨骼肌的有氧代谢能力增强，AMPKα2对CPT-1的影响，体现出了AMPKα2高表达转基因的优势。经过两周的低氧训练，转基因组的CPT-1 mRNA表达显著高于野生组（图1），并且与低氧暴露相比，低氧训练显著增加了转基因小鼠CPT-1的蛋白表达，野生小鼠却没有显著变化（图2）。说明低氧训练通过AMPKα2的高表达调节CPT-1的蛋白水平，以促进脂肪酸的氧化供能。
4 结 论
1）两周低氧和低氧训练后，AMPKα2的蛋白表达没有显著变化。
2）两周低氧暴露，显著地提高了CPT-1的mRNA表达，但对其蛋白表达没有显著作用，并且野生和转基因小鼠CPT-1的变化无差异。
3）两周低氧训练，转基因小鼠的CPT-1 mRNA表达显著高于野生小鼠，低氧训练显著增加了转基因小鼠CPT-1的蛋白表达，说明AMPKα2的高表达参与调节低氧训练中骨骼肌CPT-1的水平，进而调节脂肪酸氧化。
参考文献：
［1］ 黄熙泰,于自然,李翠凤.现代生物化学［M］.北京:化学工业出版社,2005:303-306.
［2］ Durante PE, Mustard KJ, Park SH, et al. Effects of endurance training on activity and expression of AMP-activated protein kinase isoforms in rat muscles［J］.Am J Physiol Endoc M,2002, 283(1):E178-E186.
［3］ Frosig C, Jorgensen SB, Hardie DG, et al. 5’-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle［J］.Am J Physiol Endoc M,2004,286:E411-E417.［4］ Tikkanen HO, Nfiveri HK, Harkonen MH. Alteration of regulatory enzyme activities in fast-twitch and slow-twitch muscles and muscle fibres in low-intensity endurance-trained rats［J］.Eur J Appl Physiol,1995,70:281-287.
［5］ Tunstall RJ, Mehan KA, Wadley GD, et al. Exercise training increases lipid metabolism gene expression in human skeletal muscle［J］.Am J Physiol Endoc M,2002,283:E66-72.
［6］ 张玥, 牛燕媚, 姜宁, 等. 有氧运动对C57BL/6小鼠骨骼肌PPARα及CPT-1的影响［J］. 中国运动医学杂志, 2008,27(6):690-693.
［7］ Borger DR, Gavrilescu LC, Bucur MC, et al. AMP-activated protein kinase is essential for survival in chronic hypoxia［J］.Biochem Bioph Res Co,2008,370(2):230-234.
［8］ Schneider A, Younis RH, Gutkind JS. Hypoxia-induced energy stress inhibits the mTOR pathway by activating an AMPK/REDD1 signaling axis in head and neck squamous cell carcinoma［J］.Neoplasia,2008,10(11):1295-1302.
［9］ Marsin AS, BouzinC, Bertrand L, et al. The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase［J］.J Biol Chem,2002, 277(34): 30778-30783.
［10］ Papandreou I, Lim AL, Laderoute K, et al. Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L［J］.Cell Death Differ,2008,15:1572-1581.
［11］ Jibb LA, Richards JG. AMP-activated protein kinase activity during metabolic rate depression in the hypoxic goldfish, Carassius auratus［J］.J Exp Biol,2008,211:3111-3122.
［12］ Langfort J, Viese M, Ploug T, et al. Time course of GLUT4 and AMPK protein expression in human skeletal muscle during one month of physical training［J］.Scand J Med Sci Spor, 2003, 13(3):169-74.
［13］ Nielsen JN, Mustard KJW, Graham DA, et al. 5’-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle［J］.J Appl Physiol,2003,94:631-641.
［14］ Huss JM, Levy FH, Kelly DP. Hypoxia inhibits the peroxisome proliferator-activated receptor α/retinoid X receptor gene regulatory pathway in cardiac myocytes: a mechanism for O 2 -dependent modulation of mitochondrial fatty acid oxidation［J］.J Biol Chem,2001, 276(29): 27605-27612.
［15］ Galbes O, Goret L, Caillaud C, et al. Combined effects of hypoxia and endurance training on lipid metabolism in rat skeletal muscle［J］.Acta Physiol,2008,193:163-173.
［16］ Hoppeler H, Vogt M, Weibel ER, et al. Response of skeletal muscle mitochondria to hypoxia［J］.Exp Physiol,2003, 88(1): 109-119.
［17］ Razeghi P, Young ME, Abbasi S, et al. Hypoxia in vivo decreases peroxisome proliferator-activated receptor α-regulated gene expression in rat heart［J］.Biochem Bioph Res Co,2001,287:5-10.
［18］ Heck AL, Barroso CS, Callie ME, et al. Gene-nutrition interaction in human performance and exercise response［J］. Nutrition, 2004, 20: 598-602.
［19］ Jong-Yeon K, Hickner RC, Dohm GL, et al. Long- and medium-chain fatty acid oxidation is increased in exercise-trained human skeletal muscle［J］.Metabolism, 2002, 51(4):460-464.
［20］ McClelland GB. Fat to the fire: the regulation of lipid oxidation with exercise and environmental stress［J］.Comp Biochem Phys B,2004, 139:443-460.
关键词：AMPKα2；CPT-1；低氧；低氧训练；骨骼肌
中图分类号：G804.7文献标识码：A文章编号：1007-3612(2011)01-0051-04
The Effects of Hypoxia and Hypoxia Training on the Expression of CPT1 in AMPKα2 Transgenic Mice Skeletal Muscle
YAO Lu，XIE Jin,XIE Jin，LI Songbo，ZHANG Ying
(Beijing Sport University, Beijing 100084, China)
Abstract:Objective: To study the effects of hypoxia and hypoxia training on the expressions of CPT1 mRNA and protein in AMPKα2 transgenic mice skeletal muscle. Furthermore, we explore the possible mechanism of AMPKα2 in regulating fatty acid oxidation during hypoxia and hypoxia training. Methods: Healthy C57BL/6J wildtype(WT) mice and AMPKα2 transgenic(TG) mice, two months old, were randomly divided into control groups, hypoxia groups and hypoxia training groups each.Hypoxia groups and hypoxia training groups were exposed in the hypoxia room the whole day for two weeks (simulation of 4000m altitude, oxygen concentration 12.3%), and meanwhile hypoxia training groups were trained to run on the treadmill (12 m/min, 1h/day). After that, quadriceps femoris muscle were removed, and then the expression of AMPKα2 protein and CPT1 mRNA and protein were measured. Results: Either hypoxia or hypoxia training had no effect on the expression of AMPKα2 protein; both hypoxia and hypoxia training significantly increased CPT1 mRNA expression(P<0.05); in hypoxia groups, there was no apparent difference in CPT1 expression between WT and TG mice; after two weeks hypoxia training, the expression of CPT1 mRNA of TG mice was significantly higher than that of WT mice(P<0.05) and compared with hypoxia groups, the expression of CPT1 protein of TG mice was higher in hypoxia training groups(P<0.05), but hypoxia training didn’t change that of WT mice. Conclusion: During hypoxia training, AMPKα2 is activated to regulate the expression level of CPT1 in skeletal muscle, so that it stimulates fatty acid oxidation.
Key words: AMPKα2; CPT1; hypoxia; hypoxia training; skeletal muscle
5’-腺苷酸活化的蛋白激酶（5’-AMP-activated protein kinase, AMPK）在调节和维持能量代谢平衡中发挥着非常重要的作用，被称为细胞的能量感受器。运动、缺血、缺氧等刺激都会造成细胞内的AMP/ATP比值升高，使 AMPK活化并作用于下游靶蛋白，开启ATP的合成途径，同时关闭多条ATP的分解途径。脂肪酸的β氧化是ATP再合成的重要途径之一，然而长链脂肪酸不能通过扩散的方式进入线粒体，肉碱棕榈酰转移酶-1（carnitine palmitoyl transferase1，CPT-1）位于线粒体外膜的外侧，是长链脂肪酸进入线粒体氧化的限速酶［1］。已有研究表明长期适当的运动训练能够增加AMPK和CPT-1的活性和表达水平［2-6］，但对于低氧和训练结合时AMPK和CPT-1表达水平的研究尚少。本实验旨在观察低氧和低氧训练对AMPKα2转基因小鼠骨骼肌CPT-1的mRNA和蛋白表达的影响，以探讨AMPKα2调节脂肪酸氧化的可能机制。
1 实验材料与方法
1.1 实验对象与分组 健康两月龄的C57BL/6J品系小鼠，野生小鼠（Wild-type，WT）和AMPKα2转基因小鼠（Transgenic，TG）各30只，体重(18±2)g。AMPKα2转基因小鼠由中国医学科学院实验动物研究所建立并繁育，全身高表达AMPKα2基因。所有小鼠均在北京体育大学动物房中饲养，温度20～25℃，相对湿度50°～70°，每天光照12 h，自由摄食和饮水，国家标准啮齿类动物饲料。
不同实验结果的差异性可能是由实验对象、取材部位、研究方法、低氧暴露模式（如氧浓度、低氧持续时间）等方面的原因造成的。
有学者指出长期低氧应激使骨骼肌纤维中的线粒体含量减少［16］，机体更加依赖糖类作为代谢底物［16,17］。在产生等量ATP的情况下，脂肪氧化的耗氧量多于糖代谢，所以低氧适应可能更多地通过增加糖的有氧氧化和糖酵解过程为机体提供能量［17］。因此，本实验中，低氧对野生和转基因小鼠CPT-1的作用无差异，可能与此有关。在低氧环境下AMPKα2的高表达，可能更多地体现在对糖代谢的调节上，而对脂肪氧化中的CPT-1的影响不大。目前，关于低氧对骨骼肌CPT-1影响的研究还很少，有待进一步深入研究。
3.2.2 低氧训练对AMPKα2转基因小鼠CPT-1表达的影响 运动可以促进脂肪酸氧化，改善对储备脂肪的利用，抑制脂肪组织的增长［18］。运动激活的AMPK将脂肪酸合成的限速酶——乙酰辅酶A羧化酶（acetyl-CoA carboxylase，ACC）磷酸化并抑制其活性，同时磷酸化丙二酰辅酶A脱羧酶（malonyl-CoA decarboxylase，MCD）使其活化，减少了丙二酰CoA的产生，解除对CPT-1的抑制，CPT-1的活性增强，进入线粒体的脂肪酸增多，促进β氧化。
人类肌肉活检和动物实验都发现耐力训练能够适应性地提高骨骼肌CPT-1的活性［3，4，19］。运动训练除了改善酶的活性，还可以引起表达水平的变化，对代谢的影响更为深刻［20］。每天以63%VO2max的强度蹬车1 h，连续运动9 d，CPT-1的mRNA表达显著提高［5］。张癑等发现，高脂饮食结合运动组大鼠骨骼肌中CPT-1的转录和翻译水平发生了显著的增加［6］。在本研究中，低氧训练组由于低氧和运动的双重刺激，使机体处于极大地应激状态。骨骼肌的有氧代谢能力增强，AMPKα2对CPT-1的影响，体现出了AMPKα2高表达转基因的优势。经过两周的低氧训练，转基因组的CPT-1 mRNA表达显著高于野生组（图1），并且与低氧暴露相比，低氧训练显著增加了转基因小鼠CPT-1的蛋白表达，野生小鼠却没有显著变化（图2）。说明低氧训练通过AMPKα2的高表达调节CPT-1的蛋白水平，以促进脂肪酸的氧化供能。
4 结 论
1）两周低氧和低氧训练后，AMPKα2的蛋白表达没有显著变化。
2）两周低氧暴露，显著地提高了CPT-1的mRNA表达，但对其蛋白表达没有显著作用，并且野生和转基因小鼠CPT-1的变化无差异。
3）两周低氧训练，转基因小鼠的CPT-1 mRNA表达显著高于野生小鼠，低氧训练显著增加了转基因小鼠CPT-1的蛋白表达，说明AMPKα2的高表达参与调节低氧训练中骨骼肌CPT-1的水平，进而调节脂肪酸氧化。
参考文献：
［1］ 黄熙泰,于自然,李翠凤.现代生物化学［M］.北京:化学工业出版社,2005:303-306.
［2］ Durante PE, Mustard KJ, Park SH, et al. Effects of endurance training on activity and expression of AMP-activated protein kinase isoforms in rat muscles［J］.Am J Physiol Endoc M,2002, 283(1):E178-E186.
［3］ Frosig C, Jorgensen SB, Hardie DG, et al. 5’-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle［J］.Am J Physiol Endoc M,2004,286:E411-E417.［4］ Tikkanen HO, Nfiveri HK, Harkonen MH. Alteration of regulatory enzyme activities in fast-twitch and slow-twitch muscles and muscle fibres in low-intensity endurance-trained rats［J］.Eur J Appl Physiol,1995,70:281-287.
［5］ Tunstall RJ, Mehan KA, Wadley GD, et al. Exercise training increases lipid metabolism gene expression in human skeletal muscle［J］.Am J Physiol Endoc M,2002,283:E66-72.
［6］ 张玥, 牛燕媚, 姜宁, 等. 有氧运动对C57BL/6小鼠骨骼肌PPARα及CPT-1的影响［J］. 中国运动医学杂志, 2008,27(6):690-693.
［7］ Borger DR, Gavrilescu LC, Bucur MC, et al. AMP-activated protein kinase is essential for survival in chronic hypoxia［J］.Biochem Bioph Res Co,2008,370(2):230-234.
［8］ Schneider A, Younis RH, Gutkind JS. Hypoxia-induced energy stress inhibits the mTOR pathway by activating an AMPK/REDD1 signaling axis in head and neck squamous cell carcinoma［J］.Neoplasia,2008,10(11):1295-1302.
［9］ Marsin AS, BouzinC, Bertrand L, et al. The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase［J］.J Biol Chem,2002, 277(34): 30778-30783.
［10］ Papandreou I, Lim AL, Laderoute K, et al. Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L［J］.Cell Death Differ,2008,15:1572-1581.
［11］ Jibb LA, Richards JG. AMP-activated protein kinase activity during metabolic rate depression in the hypoxic goldfish, Carassius auratus［J］.J Exp Biol,2008,211:3111-3122.
［12］ Langfort J, Viese M, Ploug T, et al. Time course of GLUT4 and AMPK protein expression in human skeletal muscle during one month of physical training［J］.Scand J Med Sci Spor, 2003, 13(3):169-74.
［13］ Nielsen JN, Mustard KJW, Graham DA, et al. 5’-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle［J］.J Appl Physiol,2003,94:631-641.
［14］ Huss JM, Levy FH, Kelly DP. Hypoxia inhibits the peroxisome proliferator-activated receptor α/retinoid X receptor gene regulatory pathway in cardiac myocytes: a mechanism for O 2 -dependent modulation of mitochondrial fatty acid oxidation［J］.J Biol Chem,2001, 276(29): 27605-27612.
［15］ Galbes O, Goret L, Caillaud C, et al. Combined effects of hypoxia and endurance training on lipid metabolism in rat skeletal muscle［J］.Acta Physiol,2008,193:163-173.
［16］ Hoppeler H, Vogt M, Weibel ER, et al. Response of skeletal muscle mitochondria to hypoxia［J］.Exp Physiol,2003, 88(1): 109-119.
［17］ Razeghi P, Young ME, Abbasi S, et al. Hypoxia in vivo decreases peroxisome proliferator-activated receptor α-regulated gene expression in rat heart［J］.Biochem Bioph Res Co,2001,287:5-10.
［18］ Heck AL, Barroso CS, Callie ME, et al. Gene-nutrition interaction in human performance and exercise response［J］. Nutrition, 2004, 20: 598-602.
［19］ Jong-Yeon K, Hickner RC, Dohm GL, et al. Long- and medium-chain fatty acid oxidation is increased in exercise-trained human skeletal muscle［J］.Metabolism, 2002, 51(4):460-464.
［20］ McClelland GB. Fat to the fire: the regulation of lipid oxidation with exercise and environmental stress［J］.Comp Biochem Phys B,2004, 139:443-460.